Force in muscle fibers is generated via conformational changes of myosin while attached to actin. These conformational changes, are coupled with ATP hydrolysis by the myosin ATPase, which provides energy for mechanical work. It has been proposed that conformational changes in muscle contraction may involve rotation of myosin heads (H. E. Huxley, 1969; A. F. Huxley and Simmons, 1971). The goal of present study was to correlate orientation and dynamics of myosin heads to biochemical intermediates of ATPase cycle using spectroscopic molecular probes. / To determine the pre-power stroke conformation of myosin heads, contractile cycle was trapped by aluminum fluoride. Orientation and dynamics of pre-power stroke myosin heads in skined muscle fibers labeled with maleimide or $\alpha$-iodoketo spin label was determined using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR respectively. In putative pre-power stroke state myosin heads are disordered as relaxed heads, but their mobility is significantly restricted. Modeling of the immobile fraction (35%), as estimated from stiffness, suggest that bound heads rotate with a correlation time of $\tau\sb{\rm r}$ = 150-400 $\mu$s, as compared to $\tau\sb{\rm r}$ = 3 $\mu$s for the relaxed heads. / In EPR study of post-power stroke states, approximated by the addition of MgADP to rigor muscle fibers, $\alpha$-iodoketo spin label attached at cys-707 of myosin head revealed that the labeled domain executes large-amplitude microsecond motions with rates $\tau\sb{\rm r}$ = 150 $\mu$s. / To induce states that occurs near the end of power stroke, positive strain was applied to muscle fiber in absence of nucleotides. EPR spectra of $\alpha$-iodoketo labeled fiber demonstrated that there is no change in orientational distribution of the probes upon application of positive strain. Similarly, application of negative strain, which was intended to model those heads which are responsible for generating load during filament sliding, did not have any effect on orientational distribution of the probes. These results indicate that the rigor bond is very stereospecific and very strong. / Source: Dissertation Abstracts International, Volume: 56-12, Section: B, page: 6613. / Major Professor: Piotr G. Fajer. / Thesis (Ph.D.)--The Florida State University, 1995.

Humans are exposed daily to the deleterious effects of acute environmental stressors. Experimental research can provide solutions to the problems of protecting human life in adverse environments. However, it is not always possible or practical to employ human subjects when exploring acute or extreme conditions. Consequently, an applied nonhuman model may provide the best substitute for human subjects to assess the environmental impact. The model must be able to be evaluated and respond validly and reliably to the stressor. / In this study, a primate model was assessed during cold stress that conceivably may be encountered by civilian or military workers. Specifically, two of the hormones responsible for fluid shifts in the body during acute cold exposure and their relationships to urine output and body temperatures were studied in five male adult rhesus monkeys (Macaca mulatta). Plasma concentrations of vasopressin and norepinephrine were measured before, during (every 30 minutes), and after 2 hours at 6 and 26$\sp\circ$C. Urine volume and sodium and potassium concentrations were also determined. The monkeys did not exhibit diuresis or natriuresis during the exposure. Plasma vasopressin and norepinephrine increased significantly (p $<$ 0.05) during cold exposure. These data suggest that both vasopressin and norepinephrine are important in the body fluid response of the rhesus monkey during acute cold stress. It appeared that vasopressin may be the dominant hormone because of the antidiuretic effect observed. Norepinephrine did not induce a diuretic effect. This response is contradictory to previous works with other animals and humans. Despite these results, the rhesus monkey appeared to be suitable for examining the effects of cold at rest under controlled environmental conditions. However, data from cold research with this primate model may not extrapolate to humans or other animals. / Source: Dissertation Abstracts International, Volume: 57-03, Section: B, page: 1549. / Major Professor: Emily Haymes. / Thesis (Ph.D.)--The Florida State University, 1996.

O Lepidoptera Diatraea saccharalis possui três &#946;-glicosidases digestivas solúveis. As três enzimas foram resolvidas através de uma combinação de passos cromatográficos em colunas Econo Pac Q ( sistema de baixa pressão), Mono Q, Phenyl Superose, Superose e Mono P (FPLC). Duas delas, denominadas de E1 e E2, foram purificadas até a homogeneidade, enquanto a terceira (E3) foi semi-purificada. Dentre todas as técnicas cromatográficas testadas, colunas hidrofóbicas são as únicas capazes de resolver as três atividades de &#946;-glicosidase. Os pesos moleculares relativos (determinados por SDS PAGE), pH ótimo e o pl foram respectivamente: E1, 58 K, 6,7, 7,5; . As três enzimas apresentam quatro subsítios de ligação de glicose e têm especificidade ampla, sendo capazes de clivar substratos sintéticos, dissacarídeos, oligossacarídeos e glicosídeos tóxicos produzidos por plantas. O principal papel fisiológico de E1 deve ser a clivagem de oligocelodextrinas derivadas da digestão de hemicelulose. E2 deve estar envolvida na hidrólise de glicolipídios, sendo a E3 a principal responsável pela digestão de dissacarídeos. / The Lepidoptera Diatraea saccharalis has three soluble digestive &#946;-glycosidases. They were resolved by a combination of chromatographic steps, using Econo Pac Q (low-pressure system), Mono Q , Phenyl Superose, Superose 12 and Mono P (FPLC system) columns. Two enzymes called E1 and E2 were purified to homogeneity. The third (E3) was semi-purified. Hydrophobic columns are the only ones able to resolve the three &#946;-glycosidase activities. The relative molecular weights (SDS PAGE}, pH optimum and pi values were, respectively: E1 , 58 K, 6.7, 7.5; E2, 61 K, 6.3, 7.4; E3, 61 K, 7.2, 7.4. The enzymes have four sub-sites for glucon binding in their active sites. They have broad substrate specificity to hydrolyze synthetic substrates, disaccharides, oligosaccharides and plant toxic glucosides. E1 may have the physiological role of hydrolyzing oligosaccharides derived from hemicellulose digestion. E2 may be responsible for glycolipid digestion and E3 seems to be the main enzyme that digests disaccharides.

The paraventricular nucleus of the hypothalamus (PVN) is the main central integrator in the neuroendocrine and preautonomic control of body homeostasis and the adaptation to stress. Glucocorticoids were shown to stimulate rapid release of an unidentified retrograde messenger in the PVN, which causes inhibition of synaptic glutamate release onto PVN neurosecretory neurons. Fasting leads to increased levels of circulating glucocorticoids and causes increased hypothalamic levels of endocannabinoids. Leptin, a primary signal of nutritional state, blocks endocannabinoid biosynthesis in the hypothalamus and suppresses CB1 cannabinoid receptor-dependent hyperphagia in fasting animals. Whole-cell-patch-clamp recordings and biochemical assays followed by liquid chromatography-mass spectrometry analysis were used in acute hypothalamic slices to test two main hypothesis: (1) that glucocorticoids rapidly stimulates the biosynthesis and release of endocannabinoids in the PVN, causing inhibition of synaptic glutamate release onto PVN neurons; and (2) that leptin blocks glucocorticoid-induced synthesis of endocannabinoids in the PVN, preventing glucocorticoid inhibitory effect. The data presented here support both hypothesis and further demonstrate that (1) glucocorticoids rapidly trigger the biosynthesis and retrograde release of the endocannabinoids anandamide and 2-arachdonylglycerol in the PVN by a mechanism that depends on the activation of a membrane receptor and a Galpha s-cAMP-PKA signaling pathway; (2) leptin blocks glucocorticoid-induced endocannabinoid biosynthesis in the PVN by triggering ob-R leptin receptor-dependent activation of phosphodiesterase 3B; and (3) leptin blocks glucocorticoid-induced, CB1 cannabinoid receptor-dependent inhibition of glutamatergic inputs to oxytocin- and vasopressin-expressing PVN magnocellular neurons, which are involved in energy homeostasis, fluid balance, hemodynamics and the stress response in male rats. This hormonal interaction is likely to mediate PVN-dependent preautonomic and neurosecretory adaptations that take place to maintain energy homeostasis during basal and stress-related conditions, including caloric deficit during fasting and satiety upon refeeding. Furthermore, the non-genomic interaction between glucocorticoids- and leptin-activated signaling pathways demonstrated here reveals a nutritional state-sensitive, endocannabinoid-mediated regulatory mechanism to modulate and coordinate the hypothalamic control of energy homeostasis, fluid balance and the stress response. Sustained elevated circulating levels of GCs (e.g., by chronic stress or depression) or leptin (e.g., by obesity) could result in deregulation of this mechanism, leading to eating, metabolic and cardiovascular disorders / acase@tulane.edu

With the interest in somatostatin as an inhibitory regulator of nutrient absorption, this study was designed to investigate changes in gastrointestinal somatostatin after a voluntary shift in diet towards increased carbohydrates and sucrose in otherwise normal rats. In the first experiment a complex 'cafeteria style' high-sucrose diet was offered to experimental animals (n = 5) for one month as an additional option to the usual lab chow and water of controls (n = 5). In the second experiment rats were offered the added option of a 32% sucrose solution for five months (n = con-24, exp-26) and thirteen months (n = con-4, exp-6). A significant increase in daily caloric intake was demonstrated with the sucrose-solution diet, where approximately two-thirds of their caloric intake was taken as sugar water. A 14% and 33% weight increase due to increased adiposity was observed in the 5 and 13 month groups respectively. Insulin response to I.V. glucose was significantly correlated to weight at sacrifice in controls but unrelated in experimental animals and was significantly lower (53-63%) in older animals. Significant increases in tissue somatostatin-like immunoreactivity (SLI) were observed in the pancreas after one and five months, and in the antral stomach after five and thirteen months. No diet effect was observed in whole brain, duodenal, jejunal or ileal SLI. Portal plasma SLI was significantly decreased after I.V. glucose relative to basal (64%) and controls (59%) in the 5m experimental rats. Multiple regression analysis was used to see which of the measured factors were predictive of the final weight of the 5m rats. While variability in insulin response plays a dominant role in the prediction of the final weight of control animals, other factors such as pancreatic and brain SLI, gain importance in animals consuming sucrose solutions. These relationships are consistent with, but do not prove, a functional role of insulin and these other factors in the regulation of weight. It is concluded that chronic high-sucrose diets can increase pancreatic and stomach SLI and decrease portal plasma SLI and a mechanism of decreased somatostatin release is postulated / acase@tulane.edu

acase@tulane.edu

The blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier are composed of specialized capillaries and astrocytic endfeet that serve to regulate the environment of the central nervous system. Peptide transport system-1 (PTS-1) is located at the BBB and transports the tetrapeptide methionine enkephalin (Met-enkephalin) from brain to blood. PTS-1 and transcription play important roles in the regulation of Met-enkephalin in vivo Met-enkephalin has been associated with the consumption of ethanol. Increasing concentrations of Met-enkephalin within the brain, either by delivering the peptide into the ventricular system or by inhibiting its degradation, reduces drinking. Opiate antagonists reduce the consumption of ethanol in laboratory animals and in humans In this work, the relationships among Met-enkephalin, PPE mRNA, and PTS-1 were studied in animal models of alcoholism. In outbred ICR mice, the consumption of ethanol results in an increase in concentrations of Met-enkephalin in whole brain. This increase correlates with a decrease in the transport of Met-enkephalin out of the brain by PTS-1. The activity of PTS-1 recovers quickly after the removal of ethanol, although it remains resistant to inhibition by serotonergic agents. Levels of PPE mRNA correlate with concentrations of Met-enkephalin in naive, but not exposed, mice. These results suggests that ethanol does not regulate concentrations of Met-enkephalin through a direct effect on transcription Withdrawal-seizure prone (WSP) and withdrawal-seizure resistant (WSR) mice serve as a genetic model for sensitivity to the effects of ethanol. In naive animals, concentrations of Met-enkephalin are greater in WSP mice than in WSR mice. This difference cannot be explained by differences in levels of PPE mRNA or in activity of PTS-1. After exposure to ethanol, the concentration of Met-enkephalin increased in WSR mice, reducing the gap between WSP and WSR mice. These change were not associated with changes in PPE mRNA or in the activity of PTS-1. Differences in the function of PTS-1 exist between WSP and WSR mice since concentrations of Met-enkephalin correlate with transport rates in WSR, but not in WSP, mice. The elevated baseline in concentrations of Met-enkephalin in WSP mice, coupled with the absence of an increase during withdrawal, suggests that WSP mice experience a novel form of enkephalin resistance / acase@tulane.edu

Little information is available concerning the role of MDCs in the kidney or their role in renal pathophysiology. Using degenerate primers for the metalloprotease and disintegrin domains of the MDC family, cDNA templates from tubules, glomeruli and glomerular epithelial cells yielded a single, 195 bp product which upon sequence analysis corresponded to the disintegrin domain of mouse MDC9. Northern analysis on the tubules, glomeruli and GECs, revealed a 3.9 kb transcript identical to mouse MDC9. Western analysis of ConcanavalinA enriched glomerular extracts revealed 116 kDa and 76 kDa proteins, which upon reduction changed to the corresponding 124 kDa and 84 kDa forms. Histochemical studies revealed a basolateral distribution of MDC9 in tubules and glomerular epithelial cells, which colocalized with the betal integrin chain Expression of MDC9-GFP fusion protein in renal epithelial cells revealed a punctate basolateral distribution. Transient overexpression of MDC9 disintegrin domain led to detachment of cultured renal epithelial cells and MDC9 disintegrin domain complexed with alpha3, alpha5 and alpha6 integrins. Overexpression of MDC9-GFP facilitated fusion of renal epithelial cells compared to control GFP expressing cells. Enrichment of rat cortical lysates, using anti-MDC9 IgGs, immunoprecipitated a 116 kDa renal gelatinase. However recombinant MDC9 metalloprotease domain lacked gelatinase activity To explore the role of MDC9 in renal pathophysiology, its expression and localization was examined in rats with experimental (puromycin aminonucleoside/PAN) nephrotic syndrome. MDC9 mRNA levels (RT-PCR) and protein levels (Western) were not significantly altered, in glomeruli and tubules, prepared from rats sacrificed at ONSET of proteinuria (urinary protein/24 hours: PAN 86 + 1.9; control = 18 + 0.8) and PEAK of proteinuria (urinary protein/24 hours: PAN 1470 + 7.6; control = 26 + 1). However immunoperoxidase chemistry revealed a gradual shift in localization of MDC9 with progression of proteinuria: from exclusively basolateral in controls; to basolateral and luminal at the onset of proteinuria; to primarily luminal at the peak of proteinuria. Similar changes in localization were also observed for the EGF receptor. These observations and the ability of MDC9 to cleave HB-EGF, a mitogenic EGF receptor ligand, suggest a role of MDC9 in renal epithelial repair and/or regeneration / acase@tulane.edu

Endocrine disruption by anthropogenic chemicals has been well documented for vertebrates but has not been extensively studied for invertebrates. However, concern has been expressed over the possibility that the same environmental agents that interfere with male development in vertebrates may also perturb male development in invertebrates, for instance, cladocerans. My study with Daphnia magna has shown that the estrogenic xenobiotics diethylstilbestrol (DES) and endosulfan do not interfere with male differentiation but do inhibit molting of daphnids. Further investigation revealed that some other estrogenic agents, such as a PCB formulation Aroclor 1242, diethyl phthalate, and 2,4,5-trichlorobiphenyl (PCB29), also delay molting of Daphnia magna To gain insight into the mechanisms for the inhibition of molting caused by these estrogenic agents, a chitinolytic enzyme, chitobiase (also known as N-acetyl-beta-glucosaminidase), was used because this chitin-degrading enzyme is at the terminal of cascades of endocrine events that control molting of crustaceans and appears to be a product of genes that are regulated by the arthropod molting hormone, 20-hydroxyecdysone. I found that activity of chitobiase in the epidermis and hepatopancreas of Uca pugilator is modulated over the duration of the molting cycle with maximum enzymatic activity in both tissues occurring in late premolt, substage D3-4. This pattern of change in chitobiase activity coincides well with changes in the hemolymph titer of ecdysteroids during the molting cycle reported for the same species. Multiple injections of 20-hydroxyecdysone into premolt substage D1 fiddler crabs elevated chitobiase activity in the epidermis and hepatopancreas. These results are the first direct evidence that chitobiase is a marker for the actions of the molting hormone in a crustacean To determine whether the delay of molting in the presence of estrotrogeic xenobiotics is linked to the inhibition of activity of chitobiase, Uca pugilator was exposed up to seven days to Aroclor 1242, DES, diethyl phthalate, endosulfan, 4-octylphenol, 4-tert-octylphenol, and PCB29. Significant declines in chitobiase activity were apparent in the epidermis and hepatopancreas of Uca pugilator. The inhibition of epidermal enzymatic activity by Aroclor 1242, DES, endosulfan, and PCB29 can at least partly account for the slowing of molting in crustaceans caused by these chemicals since chitobiase is essential for degradation of the old chitinous exoskeleton. Because activity of chitobiase is regulated by 20-hydroxyecdysone, the inhibition of enzymatic activity in the presence of various estrogenic agents strongly suggests that exposure to these compounds disturbs the Y-organ-ecdysteroid receptor axis in Uca pugilator External features of a sexually abnormal fiddler crab that carries characteristics of both sexes are described / acase@tulane.edu

This dissertation tested the hypothesis that the mammotrophic pituitary tumor/somatotroph (MtT/S) cell line is a model of the normal somatotroph which may be used to study the integrated regulation of growth hormone (GH) mRNA levels by physiologically relevant hormones. Quantification of GH mRNA levels by ribonuclease protection assay indicated that MtT/S cells are stimulated in a dose-dependent manner by treatment with either triiodothyronine or dexamethasone. Although the GH-releasing hormone (GHRH)-receptor mRNA was present in MtT/S cells, GH mRNA levels were not increased by GHRH. Furthermore, GHRH did not stimulate cyclic adenosine 3',5' monophosphate (cAMP), but cAMP was chronically elevated. Consistent with constitutive activation, GH mRNA levels were only slightly elevated by forskolin and were unresponsive to 3-isobutyl-1-methylxanthine. However, MtT/S cells did not express endogenous GHRH or a constitutively activated mutant G-protein alpha s, subunit, the most common causes of elevated cAMP in type 2 acromegalic adenomas Increasing concentrations of insulin-like growth-factor (IGF-1) reduced MtT/S cell GH mRNA levels in a switch-like/ultrasensitive manner. These kinetics were IGF-1 specific, as insulin reduced GH in a hyperbolic fashion. IGF type 1 and insulin receptors were detected by western blot. Pit-1 mRNA levels were not altered in IGF-1-treated cultures. Selective inhibitor treatment of MtT/S cells with wortmannin, LY 294,002 and PD098059 suggested that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase-dependent pathways are not required for IGF-1 effects GH mRNA quantification by in situ hybridization revealed that GH mRNA levels varied greatly among individual cells. Mathematical analysis indicated that IGF-1 does not act in an ultrasensitive manner in individual cells. Interestingly, thyroid hormone and dexamethasone diminished the ultrasensitivity of the average response to IGF-1. These data suggest that the ultrasensitivity observed in the average GH expression per cell is due to IGF-1 binding factors outside the cells In conclusion, these data suggest that MtT/S cells are a model of acromegalic type 2 adenoma cells of unknown etiology, rather than normal somatotrophs. With the clarification of the source of the elevated cAMP in MtT/S cells, it will be possible to use these cells to dissect the molecular mechanisms that lead to GH-producing adenomas / acase@tulane.edu